Spatial modulation of light to determine object length

ABSTRACT

Spatially modulated light emanating from an object moving along a flow path is used to determine various object characteristics including object length along the flow direction. Light emanating from at least one object moving along in a flow path along a flow direction of a spatial filter is sensed. The intensity of the sensed light is time modulated according to features of the spatial filter. A time varying electrical signal is generated which includes a plurality of pulses in response to the sensed light. Pulse widths of at least some of the pulses are measured at a fraction of a local extremum of the pulses. The length of the object along the flow direction is determined based on the measured pulse widths.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 14/181,530, filedFeb. 14, 2014, which is incorporated herein by reference in itsentirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with government support under contract numberW911NF-10-1-0479 (3711), awarded by the Department of Defense. The U.S.Government has certain rights in this invention.

TECHNICAL FIELD

This application relates generally to techniques for performing sampleanalysis by evaluating light emanating from the objects in a sample. Theapplication also relates to components, devices, systems, and methodspertaining to such techniques.

BACKGROUND

The present disclosure relates generally to techniques that determineobject characteristics using light emanating from the objects. Morespecifically, the techniques can use filter arrangements to transmitand/or reflect light with time variation, such as where the objects aremoving relative to the filter arrangements.

Various techniques have been proposed for using light emanating fromobjects. For example, U.S. Pat. No. 7,358,476 (Kiesel et al.) describesa fluidic structure with a channel along which is a series of sensingcomponents to obtain information about objects traveling within thechannel, such as droplets or other objects carried by fluid. A sensingcomponent includes a set of cells that photosense a range of photonenergies that emanate from objects. A processor can receive informationabout objects from the sensing components and use it to obtain spectralinformation. Additional techniques are described, for example, in U.S.Patent Application Publications 2008/0181827 (Bassler et al.) and2008/0183418 (Bassler et al.) and in U.S. Pat. No. 7,701,580 (Bassler etal.), U.S. Pat. No. 7,894,068 (Bassler et al.), U.S. Pat. No. 7,547,904(Schmidt et al.), U.S. Pat. No. 8,373,860 (Kiesel et al.), U.S. Pat. No.7,420,677 (Schmidt et al.), and U.S. Pat. No. 7,386,199 (Schmidt etal.).

Also, various flow cytometry techniques have been proposed.

SUMMARY

Some embodiments described herein relate to a system configured tospatially modulate light and to determine various characteristics ofobjects based on the spatially modulated light. The system includes aspatial filter having a plurality of mask features disposed along alongitudinal axis of the filter. A detector is positioned to sense lightemanating from at least one object moving in a flow path along a flowdirection that corresponds to the longitudinal axis of the filter. Asthe intensity of the sensed light is modulated according to the maskfeatures the detector generates a time varying electrical signalcomprising a sequence of time modulated pulses responsive to the sensedlight. The system includes an analyzer configured to measure a pulsewidth of at least some of the pulses at a fraction of an amplitudeextremum of the pulses. The analyzer determines a length of the objectalong the flow direction based on the measured pulse widths.

Some embodiments are directed to a method of determining object length.Light emanating from at least one object moving along in a flow pathalong a flow direction of a spatial filter is sensed. The spatial filterhas a plurality of mask features comprising first features alternatingwith second features along the flow direction. The first features havefirst light transmission characteristics and the second features havingsecond light transmission characteristics, different from the firstlight transmission characteristics. An intensity of the sensed light ismodulated according to the mask features. A time varying electricalsignal is generated which includes a plurality of pulses responsive tothe sensed light. A pulse width of at least some of the pulses ismeasured at a fraction of a local extremum value of the pulses. Thelength of the object along the flow direction is determined based on themeasured pulse widths.

The above summary is not intended to describe each disclosed embodimentor every implementation of the present disclosure. The figures and thedetailed description below more particularly exemplify illustrativeembodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

Throughout the specification reference is made to the appended drawingswherein:

FIG. 1 is an example embodiment of an assembly with a spatial filter,detector, and analyzer configured to determine object characteristicsbased on spatially modulated light;

FIG. 2 is a side cross sectional view of another example embodiment ofan assembly with the spatial filter positioned between the object andthe detector;

FIG. 3 is a schematic view of another example embodiment of an assemblywith an optical imaging element positioned between the object anddetector and the spatial filter positioned adjacent the detector;

FIG. 4 is a schematic view of another example embodiment of an assemblywith the optical imaging element positioned between the light source andthe detector and the spatial filter positioned adjacent the lightsource;

FIG. 5A is a flow diagram of a process for determining object lengthbased on spatial modulation of light in accordance with someembodiments;

FIG. 5B is a flow diagram of a process for determining object lengthbased on spatial modulation of light using a spatial filter having maskfeatures that change in length along the flow direction in accordancewith some embodiments;

FIG. 5C is a flow diagram of a process for determining object lengthbased on spatial modulation of light using a spatial filter having maskfeatures that are constant in length along the flow direction inaccordance with some embodiments;

FIG. 6A provides an idealized graph of light emanating from an objecthaving a circular cross section along the flow direction as the objecttraverses a light transmissive mask feature;

FIG. 6B provides an idealized graph of light emanating from an objecthaving a rectangular cross section along the flow direction as theobject traverses a light transmissive mask feature;

FIG. 7 shows a family of graphs of the intensity of emanating light fora number of mask feature lengths;

FIG. 8A is a perspective view of a portion of a system that includes aspatial filter having mask features that change in length along the flowdirection;

FIG. 8B shows the spatial filter of FIG. 8A in more detail;

FIG. 9 illustrates a process for determining object length byextrapolating a function fitted to measured data in accordance with someembodiments;

FIG. 10 illustrates a spatial modulation system that includes a spatialfilter with first and second symmetrical portions containing maskfeatures having a length that changes along the flow direction, thespatial filter used in conjunction with a light source with a Gaussianintensity distribution;

FIG. 11 is a graph of a signal generated by the detector of the systemdepicted in FIG. 10 as an object moves through the detection region;

FIG. 12 illustrates a spatial modulation system that includes a spatialfilter with mask features that have a length that is constant along theflow direction, the spatial filter used in conjunction with a lightsource with a Gaussian intensity distribution;

FIG. 13 is a graph of a signal generated by the detector of the systemdepicted in FIG. 12 as an object moves through the detection region;

FIG. 14 illustrates a technique useful for determining object lengthusing a spatial filter having periodic features of constant length alongthe longitudinal axis of the spatial filter;

FIGS. 15A and 15B are graphs used to illustrate a process using the timevarying detector signal for determining changes in velocity of an objectas the object travels in the detection region;

FIG. 16 shows a time varying detector signal having a shape indicativeof two closely spaced objects traveling along a flow path;

FIG. 17 shows a time varying detector signal having a shape indicativeof two overlapping objects traveling along a flow path;

FIG. 18 shows a time varying detector signal having a shape indicativeof two overlapping objects traveling along a flow path; and

FIG. 19 shows a time varying detector signal having a shape indicativeof three overlapping and/or closely spaced objects traveling along aflow path;

The figures are not necessarily to scale. Like numbers used in thefigures refer to like components. However, it will be understood thatthe use of a number to refer to a component in a given figure is notintended to limit the component in another figure labeled with the samenumber.

DETAILED DESCRIPTION

The embodiments described herein perform sample analysis to determinethe dimensional characteristics of an object, in particular, the length(sometimes referred to as diameter in the case of disc-shaped orspherically-shaped objects) of the object in a flow direction. Thedetermination of dimensional characteristics described herein is basedon spatially modulated light emanating from the object. In particular,the techniques disclosed herein make use of at least one spatial filter,also referred to as a mask, that can be deployed in a variety ofapplications, including analysis of system properties and/or detectionof various characteristics of analyte in a sample. In someimplementations, a non-imaging photodetector is used to generate a timevarying electrical output signal based on the spatially modulated lightallowing for compatibility with high-throughput cytometry.

Object length determination approaches described herein involve sensinglight emanating from an object moving along an enclosed, partiallyenclosed or unenclosed flow path. The sensed light is modulatedaccording to features of a mask as the object moves along the flow pathalong a flow direction of the mask. The mask includes a plurality ofmask features comprising first features having first light transmissioncharacteristics alternating with second features having second lighttransmission characteristics, different from the first lighttransmission characteristics. As used herein, the terms “first” and“second” identify mask features having differing characteristics andthese terms are not meant imply any particular order or arrangement ofthe mask features. For example, in some implementations, the first maskfeatures are substantially transparent and the second features aresubstantially opaque. At least one detector is positioned to sense lightemanating from at least one object moving in a flow path along the flowdirection. An intensity of the sensed light is modulated according tothe mask features. The detector generates a time varying electricalsignal comprising a plurality of time modulated pulses in response tothe sensed light. An analyzer measures the pulse widths of the pulses ata fraction of a local amplitude extremum of the pulses. For example, thelocal amplitude extremum may be a maximum amplitude for positive goingpulses and may be a minimum amplitude for negative going pulses. Theanalyzer determines one or more characteristics of the object along theflow direction based on the pulse widths, at least one of thecharacteristics being object length long the direction of flow.

The term “object” refers broadly to any object of interest to bedetected. In some applications, objects of interest are particles oranalytes that are relatively small, and may be microscopic in size.However, the techniques are broadly applicable to objects of any size orshape. A given object of interest may be or include one or a collectionof biological cell(s), virus(es), molecule(s), bead(s) (includingmicrobeads), droplets (e.g. oil in water), gas bubbles, or other bit(s)of matter.

Light can emanate from an object, whether through emission (e.g.radiation, fluorescence, incandescence, chemoluminescence,bioluminescence, other forms of luminescence, etc.), scattering (e.g.reflection, deflection, diffraction, refraction, etc.), or transmission,and can be sensed by the detector, e.g., a non-pixelated photodetector.Cells or particles may be treated, e.g., stained or tagged with asuitable fluorescent probe or other agent, in such a way that they emitlight or absorb light in a predictable fashion when illuminated withexcitation light. In this regard, the light emitted by a given excitedparticle may be fluorescent in nature, or it may constitute a form ofscattered light such as in the case of Raman scattering. For simplicity,the light that emanates from (by e.g., scattering, emission, ortransmission) by an object is referred to herein as “emanating light” or“light emanating.” It will be understood that the techniques,assemblies, apparatuses, systems, and methods described herein areapplicable to detecting all forms of light emanating from an object orconstituent parts thereof.

FIG. 1 is an example of an assembly 100 configured to determine objectcharacteristics, such as determining object length according to theapproaches discussed herein, based on spatially modulated light. Theassembly 100 includes a light source 112, a mask, e.g., a spatial filter126, a flow path, e.g., fluidic device 120, a detector 130, and ananalyzer 150. Optionally, the assembly 100 may include a signaltransform module 140, e.g., dedicated circuitry, software or acombination of software and hardware. The signal transform module isconfigured to convert the time varying signal to the frequency domain,for example, using a Fourier transform.

The fluidic device 120 is adapted to receive a sample of interest to beanalyzed. The sample may enter the fluidic device 120 at an inlet 121 athereof and exit the device 120 at an outlet 121 b thereof, flowinggenerally along the x-direction along a flow path 123 which may beformed between confining members 122, 124. The members 122, 124 may beor comprise plates or sheets of glass, plastic, or other suitablematerials. One or both of members 122, 124 may be a microscope slide ora microscope cover glass, or portion thereof. The members 122, 124 neednot, however, be planar in shape. For example, they may be portions of aunitary tube or pipe having a cross section that is circular,rectangular, or another shape. Other non-planar shapes are alsocontemplated. In some cases, confinement of the sample may not benecessary, whereupon one or both of members 122, 124 may be omitted. Atleast a portion of the confining members 122 and 124 is transmissive tolight. A portion of the confining member 122 is transmissive toexcitation light emitted by the light source 112 at least in anexcitation region 123 a. In that regard, light source 112 may emitexcitation light 112 a towards the flow path 123. Likewise, a portion ofthe confining member 124 is transmissive to light emanating from theobjects 105 at least in an excitation region 123 a. In that regard,objects 105 may generate emanating light 107 towards the detector 130.

In some cases, the light source 112 may comprise a conventional lightemitting diode (LED) source or a resonant cavity LED (RC-LED) source. Ifdesired, the light source may incorporate one or more filters to narrowor otherwise tailor the spectrum of the resultant output light.Whichever type of light source is selected, the spectral makeup orcomposition of the excitation light emitted by the source 112 ispreferably tailored to excite, scatter, or otherwise cause emanation oflight from at least some of the objects that may be present in thesample, as discussed further below.

The sample is depicted as containing exemplary objects 105 of varyingsizes and shapes. The objects 105 emanate light 107 in all directions(only some directions are illustrated). The objects 105 may have avariety of characteristics, some of which can be determined by theanalyzer 150 based on the emanating light 107.

The detector 130 receives time varying light emanating from the objects105 as modulated by the spatial filter 126 and generates an electricalsignal in response to the time varying light. The time variation in thelight detected by the detector 130 may be the result of interactionbetween the excitation light and an input spatial filter to createspatially patterned excitation light that illuminates the object 105.Alternatively, the time variation in the light detected by the detector130 may be the result of interaction between light emanating from theobjects 105 and an output spatial filter. In some embodiments, thedetector includes an additional optical filter arranged between thedetector and the objects. An optical filter can be particularly usefulwhen the emanating light is fluorescent light and the optical filter isconfigured to substantially block the wavelengths of the excitationlight and to substantially pass the wavelengths of the light emanatingfrom the objects.

The assembly 100 of FIG. 1 includes the spatial filter 126 (sometimesreferred to as a mask) which can be positioned in various locations.Dashed arrows 126 a and 126 b indicate some possible locations of thespatial filter 126 to modulate the emanating light and/or to modulatethe excitation light. For example, the spatial filter may be arrangedwithin the flow channel, outside the flow channel, on a confining memberof the flow channel, or may be arranged in any location relative to theobjects to cause light emanating therefrom to be modulated. In someconfigurations, indicated by arrow 126 a, the spatial filter 126 can bearranged between the flow channel 123 and the detector 130. In thisposition, the spatial filter 126 is referred to as an output spatialfilter. In other configurations, indicated by arrow 126 b, the spatialfilter 126 can be arranged between the light source 112 and the flowchannel 123. In this position, the spatial filter 126 is referred to asan input spatial filter. An input spatial filter may be adapted totransmit light emitted by the light source by varying amounts along theexcitation region 123 a of the flow channel 123. In this configuration,the input spatial filter creates patterned excitation light in theexcitation region 123 a of the flow channel 123.

According to various implementations, an input spatial filter maycomprise a physical mask including a sequence or pattern of first maskfeatures that have a first light transmission characteristic, e.g., aremore light transmissive, and second mask features that have a secondlight transmission characteristic, e.g., are less light transmissive.The input spatial filter may alternatively or additionally comprisemicro-optics or a patterned light source configured to create theexcitation pattern. The excitation pattern can be imaged and/or directedonto the excitation region 123 a using optical components for theimaging (e.g., lenses) and/or direction, (e.g., fiber optics orwaveguides). In some embodiments an output spatial filter may beutilized and arranged between the objects 105 and the detector 130 at adetection region 123 b of the flow channel.

In some embodiments, the excitation region 123 a and the detectionregion 123 b overlap. In other embodiments, there may be partial overlapbetween the excitation and detection regions or the excitation anddetection regions may be non-overlapping or multiple detection regionsand/or excitation regions may be used with various overlapping and/ornon-overlapping arrangements.

In the assembly 100 shown in FIG. 1, the output spatial filter may beadapted to interact with the light 107 emanating from the objects 105 inthe flow channel 123. In some embodiments, the output spatial filter maybe a physical mask comprising a sequence or pattern of first maskfeatures that are more light transmissive and second mask features thatare less light transmissive. In some embodiments, color spatial filtersmay be used such that first mask features of the color spatial filterhave a first light wavelength band pass characteristic and second maskfeatures that have a second light wavelength band pass characteristic.The first and second light wavelength band pass characteristics may benon-overlapping or partially overlapping in the wavelength range. Forexample, first light wavelength band pass characteristic may be passinggreen light and second light wavelength band pass characteristic may bepassing red light.

According to some embodiments of the assembly 100 that include an inputspatial filter, as an object 105 travels in the flow direction 123 c inthe excitation region 123 a of the flow channel 123, light emanatingfrom the light source 112 is alternately substantially transmitted tothe object 105 and substantially blocked or partially blocked fromreaching the object 105 as the object 105 travels along the flowdirection 123 c. The alternate transmission and non-transmission (orreduced transmission) of the excitation light 112 a along the flowdirection 123 c produces time-varying light 107 emanating from theobject 105. The time-varying light 107 emanating from the object 105falls on the detector 130 and, in response, the detector 130 generates atime-varying detector output signal 134.

According to some embodiments of the assembly 100 that include theoutput spatial filter configuration, light 112 a from the light source112 illuminates the object 105, causing the object 105 to emanate light107. As the object 105 travels in the flow direction 123 c in thedetection region 123 b of the flow channel 123, the output spatialfilter alternatively entirely or substantially blocks the light 107emanating from the object 105 from reaching the detector 130 andsubstantially transmits the light 107 emanating from the object 105 tothe detector 130. The alternate substantial transmission and blocking(or partial blocking) of the light 107 emanating from the object 105 asthe object 105 flows through the detection region 123 b produces timevarying light that falls on the detector 130. In response, the detector130 generates the time-varying detector output signal 134.

In some embodiments such as the embodiment of FIG. 1, the analyzer 150may include a signal transform processor 140 that converts thetime-varying detector output signal 134 to a frequency domain outputsignal 136 so as to provide spectral power as a function of frequency.The signal transform processor 140 is shown as part of the analyzer 150in this embodiment, but may be part of the detector in some embodimentsor may comprise separate circuitry in other embodiments. For example, insome embodiments, the signal transform processor 140 may be part of theanalyzer circuitry along with the detector.

For conversion, the signal processor 140 may use known techniques suchas discrete Fourier transform including, for example, a Fast FourierTransform “FFT” algorithm. Thus, the frequency domain output signal 136represents the frequency component magnitude of the time-varyingdetector output signal 134, where the frequency component magnitude isthe amount of a given frequency component that is present in thetime-varying detector output signal 134 or function. The Fourier signalpower is a relevant parameter or measure because it corresponds to thefunction or value one would obtain by calculating in a straightforwardmanner the Fourier transform (e.g. using a Fast Fourier Transform “FFT”algorithm) of the time-varying signal 134. However, other methods ortechniques of representing the frequency component magnitude, or othermeasures of the frequency component magnitude, may also be used.Examples may include e.g. the square root of the Fourier signal power,or the signal strength (e.g. as measured in voltage or current) obtainedfrom a filter that receives as input the time-varying detector outputsignal 134.

In FIG. 1, the time-varying detector output signal and/or frequencydomain signal are analyzed by analyzer 150. The analyzer 150 isconfigured to receive the time-varying detector output signal and/orfrequency domain signal and to determine length of the object and/orother object characteristics, such as object velocity, based upon thetime-varying detector output signal and/or frequency domain signal. Aswill be discussed subsequently, the various embodiments discussed hereinprovide examples of techniques for determining the length dimension ofthe object 105 using various mask designs and processing techniques. Asused herein, the length of the object 105 is a dimension of the object105 as measured in a direction substantially along a flow direction 123c of the flow channel 123, e.g., along the x-direction of the Cartesiancoordinate system of FIG. 1.

FIG. 2 is an enlarged schematic view of a portion of an assembly 200according to another example embodiment. The portion of the assembly 200illustrated in FIG. 2A includes a flow path, e.g., fluidic device 220, adetector 230, and a spatial filter 226. The device 220 is adapted toreceive a sample of interest to be analyzed. The sample may enter thedevice 220 at an inlet 221 a thereof and exit the device 220 at anoutlet 221 b thereof, flowing generally in a flow direction 223 c alongthe x-direction through a flow channel 223 formed between confiningmembers 222, 224. As illustrated in FIG. 2, at least one object 205 canbe disposed at a location within the flow channel 223. One or moreobjects in the flow channel 223 can have different lengths as measuredin the x-direction of the Cartesian coordinate system illustrated. Theobjects 205 can have different widths in the y-direction of theCartesian coordinate system and/or can have different thicknesses in thez-direction of the Cartesian coordinate system.

As discussed previously, the spatial filter 226 may comprise, forexample, a spatial mask. As will be discussed in greater detailsubsequently, the spatial filter 226 may have a plurality of maskfeatures 270. The mask features 270 can include first features having afirst light transmissive characteristic and second features having asecond light transmissive characteristic, different from the firstcharacteristic. For example, the first features 270 a may be regionsthat are more light transmissive and the second features 270 b may beregions that are less light transmissive. The pattern or sequence oftransmissive features 270 a and less transmissive regions 270 b define alight transmission function that changes based on the characteristics ofthe object. This transmission function may be substantially periodic, orit may instead be substantially non-periodic. The light emanating froman object is sensed by the detector 230, which is configured to generatea time-varying output signal in response to the sensed light aspreviously discussed in connection with FIG. 1.

The spatial filter 226 may be substantially monochromatic orpolychromatic as desired. In a monochromatic mask, the transmissiveregions 270 a all have substantially the same transmissioncharacteristic, and the non-transmissive regions 270 b also all havesubstantially the same transmission characteristic (but different fromthat of the transmissive regions 270 a). In a simple case, thetransmissive regions 270 a may all be clear, as in the case of anaperture, and the less transmissive regions 270 b may be opaque, as inthe case of a layer of black ink, light blocking layer, or otherabsorptive, reflective, or scattering material. Alternatively, thetransmissive regions 270 a may all have a given color or lightwavelength band pass characteristic, e.g., high transmission for lightemanating from an excited object, but low transmission for excitationlight. Alternatively, the less transmissive regions 270 b may have a lowbut non-zero light transmission, as in the case of a grey ink orcoating, or a partial absorber or reflector. In some embodiments, thespatial filter may include mask features that are opaque or less lighttransmissive alternating with first mask features that have a firstlight wavelength band pass characteristic in a first portion of the maskand mask features that are opaque or less light transmissive alternatingwith second mask features that have a second light wavelength band passcharacteristic in a second portion of the mask.

In the embodiment of FIG. 2, the spatial filter 226 is positionedbetween the objects 205 and the detector 230 and between confiningmember 224 and the detector. The light emanating 207 from the objects205 interacts with the spatial filter 226 to provide modulation of thelight that falls on the detector 230. In some embodiments, the spatialfilter may be positioned proximate to or within the flow channel.

FIG. 3 is a schematic view of another embodiment of a portion of anassembly 300 according to another example. The portion of the assembly300 illustrated includes a light source 312, a spatial filter 326, aflow path, e.g., fluidic device 320, and a detector 330. Similar to theembodiments of FIGS. 1, and 2, the device 320 includes an inlet 321 a,an outlet 321 b, a flow channel 323 having a flow direction 323 c, andconfining members 322, 324. The spatial filter 326 includes maskfeatures 370 including first mask features 370 a having a first lighttransmissive characteristic and second mask features 370 b having asecond light transmissive characteristic. In FIG. 3, the spatial filter326 is positioned between the objects 305 and the detector 330 and ispositioned remotely from the flow channel 323 immediately adjacent thedetector 330. An optical imaging element 380 such as a lens ispositioned between the objects 305 and the filter 326 and is configuredto image light from the objects 305 onto the spatial filter 326. Thelight emanating from the objects 305 and imaged by the element 380interacts with the spatial filter 326 to provide modulation of the lightsensed by the detector 330.

FIG. 4 is a schematic view of yet another embodiment of a portion of anassembly 400. The portion of the assembly 400 illustrated includes alight source 412, a spatial filter 426, a flow path, e.g., fluidicdevice 420, and a detector 430. Similar to the previously discussedembodiments, the device 420 includes an inlet 421 a, an outlet 421 b, aflow channel 423 having a flow direction 423 c, and confining members422, 424. The spatial filter 426 includes mask features 470 such asfirst features that are light transmissive features 470 a and secondfeatures that are less transmissive regions 470 b. In FIG. 4, thespatial filter 426 is positioned between the light source 412 and thefluidic device 420 containing the objects 405. As shown, the spatialfilter 426 is positioned remotely from the flow channel 423 immediatelyadjacent the light source 412.

Interaction between the output light from the light source 412 and thespatial filter 426 causes spatially modulated excitation light 412 a. Anoptical imaging element 480 is positioned between the filter 426 and theobjects 405 and is configured to image the spatially modulatedexcitation light 412 a onto an excitation region of the flow channel423. Additionally, the optical imaging element 480 may incorporate oneor more filters to narrow or otherwise tailor the spectrum of theresultant spatially modulated excitation light. The spatially modulatedexcitation light causes light 407 emanating from the objects 405 to bespatially modulated as well. The spatially modulated light emanatingfrom the objects 405 is sensed by the detector 430.

Embodiments discussed herein involve analytical approaches to determinevarious characteristics of objects in the flow path, such as thevelocity of the objects and the length of objects along the flowdirection of the flow path. FIG. 5A is a flow diagram illustrating aprocess of length determination in accordance with some embodiments. Theapproaches illustrated by FIG. 5A involve sensing 510 light emanatingfrom at least one object moving in a flow path along a flow direction ofa spatial filter. The spatial filter includes a plurality of maskfeatures comprising first features alternating with second featuresalong the flow direction, the first features having firstlight-transmission characteristics and the second features having secondlight transmission characteristics, different from the first lighttransmission characteristics. An intensity of the sensed light ismodulated according to the mask features. A time varying electricalsignal is generated 520 in response to the sensed light. The electricalsignal includes a sequence of time modulated pulses associated with themask features. A pulse width of at least some of the pulses is measured530 at a predetermined fraction of the maximum amplitude of the pulses.The length of the object along the flow direction is determined 540based on the measured pulse widths.

As an example, if the first mask features are substantially transparentand the second mask features are substantially opaque, the electricalsignal comprises a sequence of pulses of one polarity, e.g., positivegoing pulses caused by the increase in light intensity corresponding tothe clear features, alternating opposite polarity pulses, e.g., negativegoing pulses caused by the decrease in light intensity that decrease inamplitude at least partially corresponding to the opaque features. Thewidth of the positive going pulses, the negative going pulses, or both,may be measured and used for object length determination.

As illustrated by the flow diagram of FIG. 5B, in some embodiments thelength of the first and/or second mask features changes along the flowdirection of the spatial filter. For example, the mask feature lengthmay change linearly, logarithmically, randomly, or according to anypattern. The sensed light is modulated by the mask features having thechanging length. The detector senses 511 the modulated light andgenerates 521 an electrical signal in response to the sensed light. Theelectrical signal includes a sequence of positive going pulses caused bythe increase in light intensity, alternating with negative going pulsescaused by the decrease in light intensity. The pulse widths of at leastsome of the pulses are measured 531. For example, in some embodimentsthe pulse widths of one polarity, e.g., positive pulse widths, aremeasured at a fraction of the maximum amplitude of the pulses. Thelength the object along the flow path is determined. Object lengthdetermination is based on the measured pulse widths and involvesidentifying 541 a function, f(x) that fits the data set (x_(i),y_(i)),where each x_(i) is associated with an i^(th) mask feature and eachy_(i) is associated with an i^(th) measured pulse width corresponding tothe i^(th) mask feature. The object length is determined 551 byextrapolation of the function f(x). In some cases, the function may be alinear function, the slope and intercept of which fitted from themeasured data set using a least-square linear fit model, for example. Inother cases, the function may be a logarithmic or an exponentialfunction. Pulse width measurement outliers may be eliminated using astatistical technique such as random sample consensus (RANSAC).

In some spatial filter configurations, the first mask features are clear(or more light transmissive to the light interacting with the first maskfeatures) and the second mask features are opaque (or less lighttransmissive to the light interacting with the second mask features).Extrapolation of the object length can involve determining the value off(x) when a feature length of the first mask features is mathematicallyset to zero. In some implementations, the first light transmissioncharacteristic corresponds to a particular color of light anddetermining the length of the object involves determining the length ofan object having the particular color.

FIG. 5C illustrates another embodiment wherein the feature length of thefirst and second mask features is constant along the flow direction ofthe spatial filter. In some cases the length of the first features issubstantially equal to the length of the second features. Lightemanating from the objects is sensed 512 and an electrical signal isgenerated 522 in response to the sensed light. For length determination,the pulse widths are measured 532 at a fraction of the maximum amplitudeother than 50% (half maximum). At 50% of the maximum amplitude, thepulse width is substantially independent of object length. For example,the pulse widths of the positive and/or negative going pulses can bemeasured 532 at a fraction, e.g., 20% of the maximum amplitude of thepulses, or in a range of about 10% to about 40% or in a range of about60% to about 90% of the maximum amplitude of the pulses. It is generallymore difficult to accurately measure a pulse width at a very smalland/or very large fraction of the maximum amplitude due to the presenceof noise. Hence the operational measurement range typically excludes theregions of extreme fraction values, as well as the region near 50% ofmaximum amplitude where the pulse width is substantially independent ofthe object length.

In some implementations, the velocity of the objects can be determinedby measuring the pulse width at 50% of the maximum amplitude andcalculating an average of the positive and negative going pulse widthsin the pulse pairs. The velocity of the object is related to the slopeof the averages with respect to a pulse (or mask feature) number.

Optionally, analysis of the pulse widths of the positive and negativegoing pulses can be used to determine whether objects are slowing downor accelerating as they move in the flow path past the spatial filter asdiscussed below in conjunction with FIGS. 15A-15B. Optionally, analysisof the pulse widths of the positive and negative going pulses can beused to identify whether multiple objects are traveling together alongthe flow path and/or to determine the distance between the multipleobjects as discussed below in conjunction with FIGS. 16-19. In someimplementations, identification of multiple objects traveling in theflow path and/or determining the distance between multiple objects inthe flow path involves analysis of the modulation envelope of thepositive and negative going pulses. In some implementations, the lengthof the objects can be determined at least in part by the rise timesand/or fall times of the pulses. For mask features that have a lengthalong the flow direction equal to or greater than the length of theobjects, the pulses reach their maximum value when the object is fullyexposed in the mask feature. For objects traveling at about the samevelocity, shorter objects produce pulses that have a shorter rise timethan longer objects.

The upper portion of FIG. 6A illustrates an object having a circularcross section of radius r that traverses a mask feature of length d andheight >>2r. The lower portion of FIG. 6A is an idealized graph of theintensity of light emanating from the object as it traverses the maskfeature. The object of radius r is shown at two moments in time: at timet₁, immediately before the object begins its traverse across the maskfeature, and at time t₈, immediately after the object has completed itstraverse across the mask feature. At both of these times, t₁ and t₈,light emanating from the object is 0 in the idealized intensity graph,since the entire object is fully outside of the mask feature. At timest₂ and t₇, the light emanating from the object is 20% of the maximumintensity, I. At times t₃ and t₆, the object is half within and half outof the mask feature and the light emanating from the object is 50% ofthe maximum intensity, I. Between times t₄ and t₅, the object is fullyexposed in the mask feature and the intensity of the emanating light isat the maximum intensity, I. Note that although the object isillustrated as having a circular cross-section, the object may have anyshape or length along the flow direction. For example, the object mayhave an elliptical or oval cross section with the long axis of theellipse or oval lying along the flow direction. The analysis illustratedby FIG. 6A can be applied for objects having any cross sectional shapealong the flow direction, e.g., oval or elliptical. For an objecttraveling at known, constant velocity, v, and a known mask featurelength, d, the length of the object can be determined from the intensitypulse width at some fraction of the maximum amplitude. However, it willbe appreciated that according to the analysis of FIG. 6A, the pulsewidth at 50% intensity is equal to the mask feature length independentof the object length so long as the mask feature length is at leastequal to the object length (object length=2r in this example). The pulsewidth (in seconds) at 20% of maximum intensity is equal to t₇−t₂; thepulse width (in μm) at 20% of maximum intensity is equal to(t₇−t₂)·v=d+r. The pulse width (in seconds) at maximum intensity isequal to t₅−t₄; the pulse width (in μm) at maximum intensity is equal to(t₅−t₄)·v=d−−2r.

FIG. 6B is another example of the intensity profile of light emanatingfrom an object as it traverses a mask feature. The upper portion of FIG.6B illustrates an object of length f, and a mask feature of length d andheight >>2r. The lower portion of FIG. 6B is an idealized graph of theintensity of light emanating from the object as it traverses the maskfeature. The object is shown at two moments in time: at time t₁,immediately before the object begins its traverse across the maskfeature, and at time t₈, immediately after the object has completed itstraverse across the mask feature. At both of these times, t₁ and t₈,light emanating from the object is 0 in the idealized intensity graph,since the entire object is fully outside of the mask feature. At timest₂ and t₇, the light emanating from the object is 20% of the maximumintensity, I. At times t₃ and t₆, the object is half within and half outof the mask feature and the light emanating from the object is 50% ofthe maximum intensity, I. Between times t₄ and t₅, the object is fullyexposed in the mask feature and the intensity of the emanating light isat the maximum intensity, I.

For an object traveling at known, constant velocity, v, and a known maskfeature length, d, the length of the object can be determined from theintensity pulse width at some fractions of the maximum amplitude.However, it will be appreciated that according to the analysis of FIG.6B, the pulse width at 50% intensity is equal to the mask feature lengthindependent of the object length so long as the mask feature length isat least equal to the object length (object length=l in this example).The pulse width (in seconds) at 20% of maximum intensity is equal tot₇−t₂; the pulse width (in μm) at 20% of maximum intensity is equal to(t₇−t₂)·v≈d+½l. The pulse width (in seconds) at maximum intensity isequal to t₅−t₄; the pulse width (in μm) at maximum intensity is equal to(t₅−t₄)·v=d−l.

FIG. 7 shows a family of curves of the intensity of the light emanatingfrom an object of circular cross section with radius r, wherein theobject traverses mask features of length, d, where d is expressed as afunction r. The family of curves indicates that where d is greater thanor equal to the object length (d=2r in the case of a circular crosssection area object), the pulse width (in μm) at 50% maximum is equal tothe mask feature length. For d less than the object length, the maximumintensity decreases causing the pulse width at 50% maximum intensity todecrease. In this situation, the above relationship is no longerapplicable.

In general, for length determination by linear curve fit andextrapolation, two or more mask first features (more light transmissivefeatures) having differing lengths can be used. These mask features canbe arranged in any order, but mask features with length that varieslinearly along the flow direction, as depicted in FIG. 8A can makeanalysis more straightforward. FIG. 8A shows a perspective view of aportion of a fluidic device 820 and a spatial filter 826. The fluidicdevice 820 includes a flow channel 823 having a flow direction 823 a andconfining members 822, 824, 827, and 828. Although the confining members822, 824, 827, and 828 are positioned to define the flow channel 823, inother embodiments one or all of the confining members 822, 824, 827, and828 may not be used. The flow direction 823 a aligns generally with thex-direction of the Cartesian coordinate system illustrated in FIG. 8A.In the embodiment shown, the spatial filter 826 is disposed proximate toconfining member 822. In other embodiments, the spatial filter 826 maybe disposed within the flow channel 823, mounted to any of the confiningmembers 822, 824, 827, 828, positioned relative to any of the confiningmembers 822, 824, 827, 828, positioned on or relative to the lightsource (not shown) or detector (not shown). The detector may bepositioned in any appropriate location to sense light emanating fromobjects moving in the flow channel 823 that is modulated by filter 826.

In FIG. 8A, the spatial filter 826 is arranged in the x-y plane of theCartesian coordinate system. The spatial filter 826 can have a pluralityof mask features 870 arranged such that the modulated light from theobject and the output electrical signal that results therefrom providestime modulated pulses from which the length of the objects passingthrough the flow channel 823 can be determined. The spatial filter 826can have pattern of mask features such that a length of the features inthe x direction changes linearly, as shown in the perspective view ofFIG. 8A and the top view of FIG. 8B. The frequency (also referred to aspitch) of the mask features may be constant along the flow direction.The pitch used for the spatial filter depends on the length of theobjects being measured. For example, in some configurations, the fixedpitch may be about 30 μm. In some embodiments the pitch value may befixed for all mask features regardless of the mask feature length inorder to provide robust detection, in the frequency domain, of thepresence of an object in the channel, even when the amount of lightemanating from the object is very dim. In other embodiments, the pitchvalue may be variable between the mask features.

In the exemplary embodiment shown in FIGS. 8A and 8B, the mask features870 include first mask features 870 a that have a first lighttransmission characteristic, e.g., are more light transmissive,alternating with second mask features 870 b that have second lighttransmission characteristics, e.g. are less light-transmissive. Thefirst light transmissive characteristics of the first mask features 870a are different from the second light transmissive characteristics ofthe second mask features 870 b. As will be discussed subsequently, thelength of the first mask features 870 a and/or the length of the secondmask features 870 b can change along at least a portion of the spatialfilter 826 in the flow direction of the flow channel 823 (thex-direction in FIGS. 8A and 8B). The length of a mask feature ismeasured along the flow direction (the x-direction in FIGS. 8A and 8B).In the exemplary embodiment of FIG. 8A, the length of the first maskfeatures 870 a linearly decreases along the flow direction 823 a of theflow channel 823. The length of the second mask features 870 b linearlyincreases along the flow direction 823 a of the flow channel 823.

FIG. 8B is a plan view of spatial filter 826 of FIG. 8A. FIG. 8Billustrates the mask features 870 in greater detail. Mask features 870include first mask features 870 a alternating with second mask features870 b. In the embodiment shown, the first mask features 870 a have aconstant frequency and changing length along the x-direction. Theconstant frequency results from center-to-center distances D₁ in thex-direction that remain constant for each mask feature 870 a. Thus, eachof the first mask features 870 a has a center that is spaced a samedistance D₁ from the center of an adjacent first mask feature 870 a.Similarly, each of the second mask features 870 b has a center that isspaced a same distance D₂ from the center of an adjacent second maskfeature 870 b. In the embodiment of FIGS. 8A and 8B, D₁ is equal to D₂.Although the examples provided refer to first and second mask featureshaving first and second light transmission characteristics,respectively, it will be appreciated that a spatial filter may includeadditional third, fourth, etc. mask features, wherein each of the first,second, third, fourth, etc. mask features have different lighttransmission characteristics.

The changing duty cycles of the first and second mask features 870 a,870 b is the result of changing lengths L₁, L₂ along the x-direction.Thus, each of the first mask features 870 a has a length L₁ measuredfrom a first starting edge to a second trailing edge. The length L₁ ofthe first mask features 870 a is a function of position along the flowdirection 823 a.

In the embodiment shown, mask features 870 are patterned in a desiredmanner with dimensions D₁ and D₂ being the same and L₁ and L₂ changingin a linear manner. However, in other embodiments mask features 870 maybe patterned in another manner (e.g., quadratically, logarithmically,exponentially, inverse proportionally, and/or random) that allows for adata set of pulse widths from the output signal that are associated withlengths of mask features. Thus, the mask features of the spatial filtercan be arranged in any order, so long as a data set comprising pulsewidths as a function of mask feature length can be obtained foranalysis.

FIG. 9 illustrates a process of determining the object length using amask 970 with first mask features 970 a 1-970 a 6 that decrease linearlyin length along the flow direction. The top portion of FIG. 9 shows sixfirst mask features 970 a 1-970 a 6 that are more light transmissivealternating with five second mask features 970 b 1-970 b 5 that are lesslight transmissive. The length of the first mask features 970 a 1-970 a6 decreases linearly along the flow direction (x-direction). Feature 970a 1 has a length d+5a, where d is the length of the smallest first maskfeature 970 a 6 and a is any constant. Feature 970 a 2 has length d+4a;feature 970 a 3 has length d+3a; feature 970 a 4 has length d+2a,feature 970 a 5 has length d+a, feature 970 a 6 has length d. In thisembodiment, the pitch is constant throughout the mask, as can be seen bythe distance between the centers of subsequent mask features of eitherthe first or second mask features.

As an object moves relative to the spatial filter along the flowdirection, the emanating light is sensed by the detector (not shown inFIG. 9) which generates time modulated pulses responsive to the sensedlight. The pulses have pulse widths related to the mask feature lengthsas previously described in connection with FIG. 6A. In one scenario,using 20% of maximum intensity, the pulse width (in units of length) ofthe pulse generated as the object traverses feature 970 a 1 is d+5a+r;the pulse width of the pulse generated as the object traverses feature970 a 2 is d+4a+r; the pulse width of the pulse generated as the objecttraverses feature 970 a 3 is d+3a+r; the pulse width of the pulsegenerated as the object traverses feature 970 a 4 is d+2a+r; the pulsewidth of the pulse generated as the object traverses feature 970 a 5 isd+a+r; and the pulse width of the pulse generated as the objecttraverses feature 970 a 6 is d+r. It will be appreciated that the actualpulse widths will be measured in a range around these values due tomeasurement error and noise. The pulse width measurements provide a setof mask feature measurement points {p_(i)}, each point given byp_(i)=(xi,yi), where each x_(i) is the i^(th) mask feature length andeach y_(i) is the pulse width measurement, e.g., at 20% of the maximumvalue corresponding to the i^(th) mask feature.

The set of mask feature length measurement points {p_(i)} areconceptually shown as the set of circled points in FIG. 9. If themeasurement errors are small, the set of points would ideally fall onthe linear curve 901. In practice, however, each point may fall slightlyabove or below the curve in the y direction, due to measurement errorand noise associated with the i^(th) mask feature pulse widthmeasurement. A function f({p_(i)},x) is determined that fits the set ofpoints {p_(i)}, such as the line 901 shown in FIG. 9. The functionf({p_(i)},x) predicting the expected pulse width measurement of anhypothesized mask feature of length x, based on all the given maskfeature measurements {p_(i)}, for any value of x, not necessarilyrestricted to any one of existing feature lengths on the mask. Forexample, f({p_(i)},x) may be determined by a linear regression model,such as by using a least squares approach.

The function f(x) transforms the discrete set of pulse width measurementpoints at the given mask features lengths {p_(i)} into a continuousfunction that virtually predicts the estimated pulse width for any maskfeature length x, even if this feature length is not actually present asone of the existing mask features (i.e., the mask does not actuallyinclude a mask feature having this length). The function f({p_(i)},x)allows to extrapolate the predicted pulse width for any mask featurelength x, and in particular, for an infinitely small x→0 feature length.Extrapolating the function by mathematically setting the mask featurelength to zero effectively eliminates the mask feature length,regardless of its actual size, and yields the estimated radius of theobject, where the length of the object is twice the estimated radius.The extrapolation projects the imaginary extension 901 a of the fittedline f(x) 901 to the point where d=0 which is the virtual zero openingmask feature width. The length estimation provided by the extrapolationusing this technique is self-calibrating, i.e., does not require aseparate calibration process for each different mask, since theextrapolated function f(x) is no longer dependent on the actual lengthof the smallest mask feature size d. However, the absolute object lengthmeasurement is dependent of the velocity of the object which is assumedto be constant. The technique is well suited for measuring the objectlengths of variable object sizes, small and large, which may betraveling at different velocities in the channel because there areseveral ways to measure the particle velocity.

FIG. 10 shows a cross section of a spatial filter 1026 including thefirst half region 1074 a and second half region 1074 b arranged inrelation to a light source 1012, fluidic device 1020, and a detector1030. The spatial filter 1026 includes mask features including a numberof first mask features 1070 a that are more light transmissive and anumber of second mask features 1070 b that are less light transmissive.Objects 1005 move in the fluidic device 1020 along a flow direction 1023c and emanate light 1007. Although the objects 1005 are illustrated ashaving a length in the x direction greater than the lengths of the maskfeatures 1070, it will be understood that the lengths of the objects1005 may actually be smaller than at least some of the lengths of themask features 1070. Furthermore, it will be understood that the speed ofthe objects 1005 is substantially constant. FIG. 10 shows an intensitydistribution 1000 of light 1012 a emitted from a light source 1012 anddistributed along the flow channel 1023 of the fluidic device 1010. Inthe embodiment shown, the intensity distribution 1000 of the light 1012a is not uniformly distributed along the flow channel but rather has anapproximately Gaussian distribution, with the strongest intensity at thecenter and tapering off to either side. However, in other embodimentsthe intensity distribution may vary from the example embodimentillustrated.

In a representative embodiment, the mask features are disposed in afirst section arranged in a first linear chirp pattern and a secondsection arranged in a second linear chirp pattern, wherein the firstpattern and the second pattern are symmetrical around a center lineextending laterally across the spatial filter. The first mask featuresare substantially transparent and the second mask features aresubstantially opaque. The substantially transparent features have alength of about 1 μm at the center line of the mask. The clear featuresof the first pattern have a linear decrease in length of about 1.5 μmalong the flow direction and the clear features of the second patternhave a linear increase in length of about 1.5 μm along the flowdirection, while the pitch is constant throughout the mask at about 40μm. It should be appreciated that the above dimensions are designed fordetecting and measuring a specific range of object sizes traveling at aspecific velocity range in the channel, and will generally vary based onthe desired object size and velocity range.

FIG. 11 is a simplified plot of an electrical signal 1199 that isgenerated by detector 1030 in response to sensing the modulated lightthat has passed through the mask features of the spatial filter 1026 ofFIG. 10. The morphology of the electrical signal 1199 generated by thedetector 1030 results from the intensity distribution 1000 of the lightoutput 1012 a from the light source 1012 and the interaction of thelight 1012 a with the mask features 1070, and object specificcharacteristics. As shown in FIG. 11, the output electrical signal 1199generated by the detector 1030 includes a first set of positive goingpulses having a pulse widths (or duty cycle) that decrease with respectto time (corresponding to the first portion 1074 a of mask 1070) and asecond set of positive going pulses having pulse widths (or duty cycle)that increase with respect to time (corresponding to the second portion1074 b of mask 1070). The pulse widths are narrower at the mask center,and gradually grow wider toward either end of the mask, in accordancewith the mask pattern 1074 a and 1074 b in FIG. 10. The pulse frequencyin this example is constant and is associated with the constant pitchand the constant velocity of the object as it moves along the flow. Thepulse widths are associated with the velocity and the length of theobject.

As shown, the amplitude of the pulses in the output electrical signal1199 is initially lower toward at time t=0 due to the distribution ofthe input light 1012 a (as exhibited by intensity profile 1000, whichhas a lower intensity toward the edges 1026 a, 1026 b of the spatialfilter 1026. The amplitude of the pulses increases for a time period dueto the increase in the intensity of the input light 1012 a (asillustrated by intensity profile 1000) before falling in region 1180 dueto the decreased mask feature length of the more light-transmissiveregions 1070 a (FIG. 6A) in the center region of the spatial filter 1026which corresponds to region 1180 of the electrical signal 1199. Due tothe symmetry of the input light 1012 a and the mask pattern 1074 a and1074 b around the mask center, the electrical output signal is alsoroughly symmetric around the mask center. The amplitude of the outputelectrical signal 1199 initially increases in the time period after theregion 1180 due to the gradual increase in the mask feature length ofthe more light-transmissive regions 1070 a (FIG. 6). After increasingfor a time period, the amplitude of the output electrical signal 1199eventually decreases and finally becomes zero due to a decrease inintensity of light as shown by intensity profile 1000. The dual portionmask shown in FIG. 10 is particularly useful to increase signal to noiseratio (SNR) in the signal when a light source having a Gaussiandistribution is used because the mask features are largest where theintensity of light is smallest and the mask features are smallest wherethe intensity of light is greatest.

In addition, a particularly dim object may not generate a substantialamount of emanating light to be detectable through the narrowest firstmask features 1070 a (FIG. 6), in which case the first one or more timemodulated pulses at the center of the mask may be missing. The dualportion mask design is particularly useful for identifying missing pulsepeaks at the center of the mask, based on the constant pitch. A missingpulse is readily recognized by a resulting wider time gap betweensuccessive pulses. If instead the narrow first mask features were to beplaced at the ends of the mask, then it would be much more difficult totell if any pulses may be missing, and how many.

An analyzer can be configured to receive the output electrical signal1199, determine widths of the pulses, fit a function, e.g., a line, tothe pulse widths with respect to the lengths of the mask features 1070,and extrapolate a length of the object in the flow channel from theline. For the symmetrical dual portion mask shown in FIG. 10, themeasurement of the pulse widths reveals two data sets: (x_(i), y_(1i))and (x_(i), y_(2i)) where x_(i) corresponds to the mask feature lengths(or mask feature number) for the first portion of the mask and y_(1i)corresponds to the measured pulse widths×object velocity (in μm)produced by interaction of light with the mask features of the firstmask portion 1074 a, and y_(i2) corresponds to the pulse widths×objectvelocity (in μm) produced by interaction of light with the mask featuresof the second mask portion 1074 b. Determining the length of the objectcan involve fitting f(x) to both data sets (x, y_(1i)), (x, y_(2i)) andextrapolating the object length as previously discussed in connectionwith FIG. 9.

Some embodiments involve the use of a spatial filter wherein the lengthof the first and second features of the spatial filter is constant alongthe flow direction. In some cases the length of the first features issubstantially equal to the length of the second features. FIG. 12 is aside view of a spatial filter 1226 that includes constant lengthfeatures 1270 a, 1270 b arranged in relation to a light source 1212,fluidic device 1220, and a detector 1230. The spatial filter 1226includes mask features including a number of first mask features 1270 athat are more light transmissive and a number of second mask features1270 b that are less light transmissive. At least one object 1205 movesin the fluidic device 1220 along a flow direction 1223 c and emanateslight 1207. Although the object 1205 is illustrated as having a lengthin the x direction greater than the lengths of the mask features 1270 a,1270 b, it will be understood that the length of the object 1205 mayactually be smaller than at least some of the lengths of the maskfeatures 1270 a, 1270 b.

FIG. 12 shows an intensity distribution 1200 of light 1212 a emittedfrom a light source 1212 and distributed along the flow channel 1223 ofthe fluidic device 1210. In the embodiment shown, the intensitydistribution 1200 of the light 1212 a is approximately Gaussian.However, in other embodiments the intensity distribution may vary fromthe example embodiment illustrated.

FIG. 13 is a plot of an electrical signal 1399 that is generated bydetector 1230 in response to sensing the modulated light emanating froman object 1205 moving along the flow path. The morphology of theelectrical signal 1399 generated by the detector 1230 results from theintensity distribution 1200 of the light output 1212 a from the lightsource 1212 and the interaction of the light 1212 a with the maskfeatures 1270 a, 1270 b, and object specific characteristics. As shownin FIG. 13, the output electrical signal 1399 generated by the detector1230 includes a first set of positive going pulses of increasingamplitude having pulse widths (or duty cycle) that are substantiallyconstant with respect to time and a set of negative going pulses ofdecreasing amplitude having pulse widths (or duty cycle) that aresubstantially constant with respect to time and equal to the pulsewidths of the positive going pulses. The pulse frequency in this exampleis constant and can be used to determine the constant velocity of theobject as it moves along the flow direction. The pulse widths of theelectrical signal 1399 are a function of the velocity and the length ofthe object.

As shown, the amplitude of the positive going pulses in the outputelectrical signal 1399 is initially low between time t=0 and t=300 dueto the distribution of the input light 1212 a, as exhibited by intensityprofile 1200, which has a lower intensity toward the edges 1226 a, 1226b of the spatial filter 1226. The amplitude of the pulses increases fora time period due to the increase in the intensity of the input light1212 a (as illustrated by intensity profile 1200) before falling due tothe decrease in the intensity of the input light 1212 a.

A spatial filter pattern wherein the length of the first mask featuresd1 and the length of the second mask features d2 are both constant alongthe flow direction, where d1 may or may not be the same as d2, is calleda periodic mask. In a periodic mask, the basic pattern is that of aperiodically repeating identical cell units, where each cell unit iscomprised of a pair of mask features: a first mask feature of length d1,followed by a second mask feature of length d2. In some approaches, aperiodic mask where all the mask openings are the same (same width andheight) may be used to determine object length along the flow direction.

The graph shown in FIG. 14 illustrates a technique for lengthdetermination of an object using a periodic mask. To measure the objectproperties such as the object length and velocity, a pulse widthmeasured at a fraction 50% intensity can be used to measure the objectvelocity (because travel time of the object traversing a mask at 50%intensity is independent of the object dimensions, and is dependent thevelocity of the object and the length of the mask feature).Simultaneously and independently of the object velocity measurement, theobject length can be measured using the pulse widths away from the 50%maximum intensity (for example, at 20% maximum or minimum intensity)during “open” and “close” times, where the open times correspond topulses that are generated when the emanating light from the objectpasses through a transparent mask feature and the close times correspondto pulses that are generated when the emanating light from the object isblocked by an opaque mask feature. Note that the pulses widths aremeasured in terms of time it takes the object to traverse an open(transmissive) or closed (opaque) mask feature.

In FIG. 14, curve 1401 plots the pulse widths at 20% maximum of the(positive) amplitude for the open features (positive pulses); curve 1402plots the pulse widths at 20% minimum of the (negative) amplitude forthe closed features (negative pulses); curve 1403 plots the averages ofthe pulse width values of curves 1401 and 1402.

In a scenario where (1) all the mask openings are identical, i.e., d1 isequal to d2; (2) the illumination is the same for each opening; and (3)the velocity is constant, the sum of each successive pair of open andclose times should remain approximately the same. However, in manyimplementations, the three conditions listed above are not met. Due tothe uneven (approximately Gaussian) light distribution on the spatialfilter as shown in FIG. 12, the openings near the center of the maskreceive much more light than the openings near the ends. The openingsnear the center are therefore the most accurate, and the particle lengthestimation accuracy deteriorates as the amount of illumination isreduced away from the center. Hence it is desirable to give more weightto the measurements near the center of the mask (with weights thatroughly approximate the illumination profile).

In addition, there may be defect in one or more of the mask features orthe fluidic device. Defects may occur, for example, if the laser used tocut the mask features may leave a ragged edge in one of the openings orif an opening got slightly covered during the manufacturing process.Hence the one defective mask feature may yield an erroneous measurementfor the one defective feature. A defective mask feature is likely thecause for the obvious drop in measured open time for peak 14 in thegraph of FIG. 14. However, the approaches discussed herein allow foridentification and elimination of such defective measurements withlittle loss in overall accuracy.

Two measurements are taken from each open and close mask feature pair,e.g., the width at 20% of the maximum intensity for the positive goingpulses and the width at 20% of the minimum intensity width for thenegative going pulses. A simple average of the measurements may producea suboptimal length estimate due to the non-uniformity of the lightprofile. Accordingly, in some implementations weighted curves are fit tothe measured open and closed feature pulse widths, with weightscorresponding to the illumination profile.

The points closer to the mask center (near peak 10, circled in the graphof FIG. 14) are given considerably more weight than the points at theends. The fitted curves 1401 a, 1402 b, are illustrated near the centerpeak. The fitting also includes an algorithm to eliminate any mask/chipdefects measurement points (such as RANSAC).

In FIG. 14, curve 1401 plots the pulse widths at 20% maximum of the(positive) intensity for the open features (positive pulses) and curve1402 plots the pulse widths at 20% minimum of the (negative) intensityfor the closed features (negative pulses). Curve 1401 a plots theweighted fitted curve for the pulse widths for the positive pulses (openfeatures) and curve 1402 a plots the weighted fitted curve for the pulsewidths for the negative pulses (closed features). Using the fittedcurves 1401 a, 1402 a, an adjusted measurement of the open and closepulse width is computed, shown at peak 10 of FIG. 14. This adjustmentslightly moves the measured points (e.g., from circle 1401 b to circle1401 c for the open pulse width). Similarly, the measured point at peak10 for the negative pulse widths 1402 is adjusted using the fitted curve1402 a. The object length is estimated from the adjusted open and closetimes. Note that the estimation of object length is based on all theavailable measurements, even though it is conceptually illustrated atpeak 10 in FIG. 14.

For each opening, the average of open and close pulse widths correspondsto a point midway between open and close values, shown as points alongcurve 1403, the curve connecting the average points is the average curve1403. Each average point on curve 1403 is (close time+open time)/2.Thus, twice the average value is the sum of (close time+open time),which should be roughly constant if the velocity is constant. Hence theaverage curve 1403 for an object that travels at a constant velocitythrough the channel should look like a horizontally flat line). In FIG.14, the average curve 1403 is increasing over time, with a positiveslope, which is an indication that the object is slowing as it travelsacross the spatial filter. The sloping average curve 1403 is the resultof the situation that arises when the object takes successively moretime to cross subsequent mask openings, on average. The slope of theaverage curve 1403 can therefore be used to tell whether the object isactually slowing or accelerating as it travels through the channel. Inaddition to the average object velocity information obtained from eitherthe time or frequency domain signals, the spatial filter can be used toprovide information about the instantaneous velocity of an object as ittravels along the flow path, including whether the object isaccelerating or slowing down, and by what amount, on a maskfeature-by-feature basis.

The changes in velocity can also be visually demonstrated by flippingthe signal 180 degrees and aligning the first and last minima pointswith the original signal to demonstrate that the peak centers do notalign up (another indication of the object slowing), as discussed inconnection with FIGS. 15A and 15B.

If multiple pulse width measurements are made for determining multipleobject characteristics, e.g., both object length and velocity, the pulsewidth at two fractional values of the maximum (for positive pulses) orminimum (for negative pulses), e.g., 20% and 50% intensity can besimultaneously measured by setting two intensity thresholds, measuringfour successive time points for each feature, and individually pairingthe 20% and 50% points together.

FIGS. 15A and 15B illustrate another process that may be implemented,e.g., by analysis circuitry 151 in FIG. 1. The process of FIGS. 15A and15B uses the time varying signal from the detector to identify changesin an object's velocity as it traverses a detection region in a systemhaving a spatial filter with constant length features. Graph 1501illustrates a time varying signal generated by the detector as an objecttraverses the detection region. The positive peaks of time varyingsignal 1501 form an upper envelope that is Gaussian shape due to theGaussian distribution of the light source that provides input light tothe system.

Changes in the velocity of the object along the flow path can bedetected by inverting the time varying signal 1501 along both they and xaxes y, forming inverted signal 1502. The distances betweencorresponding lower (or upper) peaks indicates that the object'svelocity was changing. The distances can be used to determine the amountof velocity change as the object moves through the detection region. Inthe example provided by FIGS. 15A and 15B, the increasing offsets 1511,1512, 1513, 1514 between corresponding lower peaks indicate that ittakes more time for the object to reach successively further away peaks,hence the object velocity is decreasing (i.e., object is slowing down)as it moves through the detection region and the amount of the offsets1511, 1512, 1513, 1514 can be used to determine the instantaneousvelocity of the object at any given time and/or an amount of thevelocity decrease.

FIGS. 16-19 are screen captures of system output that illustrateprocesses that may be implemented, e.g., by analysis circuitry 151 inFIG. 1, to provide additional information about multiple objects thatare in close proximity or that overlap along the flow direction as theymove through the detection region. FIGS. 16-19 illustrate several casesof multiple particles in the detection region, where the particles aretraveling separately (at some distance from each other), partiallyoverlapping, or actually touching (aggregated) and moving together. Thesystem may identify how the particles are moving together and the numberof particles based on the wave shapes. For separate non-overlappingparticles the system may independently determine the length of eachparticle. For example, as illustrated in FIG. 18, the system mayidentify that there are two particles in the detection area and that thefirst particle is traveling at a faster velocity than the secondparticle because the distance between peaks at t=400 is appreciablysmaller than at t=800.

The processes illustrated by graphs 16-19 rely on analysis of the timevarying signal generated by the detector. According to these processes,the shape of the time varying signal is analyzed to identify multipleobjects overlapping or in close proximity in the detection portion ofthe flow path. Each of the graphs have an upper modulation envelopeformed by the positive going peaks and a lower modulation envelopeformed by the negative going peaks. The software algorithm detectsmultiple particles based on criteria described below and displays therecognition of multiple particles can display a text output such as“multi-particle” in the user interface display, as can be discerned inFIG. 16-19. In some implementations, discerning multiple objects in thedetection region is performed by analyzing the upper and/or lowermodulation envelopes of the graphs.

FIG. 16 shows a graph 1600 that has the characteristic shape indicatingtwo objects flowing together in the detection region. Modulated lightemanating from the first object creates portion 1601 of graph 1600 andmodulated light emanating from the second object creates portion 1602 ofgraph 1600. The upper modulation envelope of graph 1600 exhibits twodistinct peaks 1603, 1604 indicating the presence of two closely spacesobjects. The distance between the first and second object is larger thanthe detection region, whereby the second object enters the detectionregion shortly after the first object has exited the detection region.The two particles travel in tandem, with the second object closelyfollows the first object in the flow path, but the objects are notoverlapping, as indicated by the decrease to nearly zero of the uppermodulation envelope between first 1601 and second 1602 graph portions.The first and second objects have approximately the same length alongthe flow direction as can be determined based on the approximately equalpulse widths of the graph 1600 in the first and second portions 1601,1602. Even though a portion of the second object signal falls within thedetection window in the frequency domain, the system can stilldistinguish from the time domain analysis that there are two objects inthis case. In contrast, systems that lack a spatial filter mask asdisclosed herein are prone to underestimating the number of objects bycounting only one object instead of two in case of collisions (i.e.,multiple particles in the detector region).

FIG. 17 illustrates a graph 1700 of the time varying detector signalhaving a shape indicative of two objects of approximately the samelength that are overlapping in the detection region as they travel alongthe flow path. The upper envelope of graph 1700 includes two peaks 1701,1702 corresponding to light emanating from the first and second objects,respectively. Light emanating from the first object that is detectedconcurrently with light emanating from the second object causes thelower modulation envelope of graph 1700 to be highly modulated. Thesecond object enters the detection region shortly after the firstobject, and is slightly brighter that the first object as can beappreciated from the peak envelope 1702 being larger than 1701.Furthermore, the first and second objects are traveling at slightlydifferent velocities. Initially the second object is in phase with thefirst object for the first few mask features, namely, the first andsecond objects enter and exit different mask features at roughly aboutthe same time. Hence the two waveforms closely overlap, and the lowerpeak values are close to zero when the two objects are bothsimultaneously behind the less transmissive mask features. However, oneobject is traveling slightly faster than the other. Over time, thedistance between the objects slowly changes such that the objects beginto go out of phase, i.e., one object enters a less transmissive maskfeature while the other object enters a more transmissive mask feature.In the latter case, there is always at least on object visible through aless transmissive mask feature, and in consequence there is always someemanating light reaching the detector from either one of the objects.Hence the lower peaks of graph 1700 do not return to zero. The rate atwhich the objects go in and out of phase is dependent on the differencein their velocities. The closer the difference in object speed is, theslower the rate of going in and out of phase, and the longer time itwould take to make the transition. In the case of FIG. 17, it takesalmost up to t=600 before the objects are maximally out of phase, afterwhich the particles begin to go back in phase, until they are finallyback in phase by time t=750. The difference in object length andbrightness also play a role in the resulting waveforms, although themost visible attribute is the interference pattern generated by the twoobjects traveling at slightly different speeds.

FIG. 18 illustrates a graph 1800 of the time varying detector signalhaving a shape indicative of two objects of approximately the samelengths that are overlapping in the detection region. Graph 1800 has afirst portion 1801 that is predominantly caused by light emanating fromthe first object and a second portion 1802 that is predominantly causedby light emanating from the second object. The situation is similar tothe case depicted in FIG. 17. However, in FIG. 17 the two objects aretraveling at nearly the same velocity, only slightly different. Incontrast, the objects in FIG. 18 are traveling at appreciably differentspeeds. The different object velocities are indicated by the differentpulse widths in the first 1801 and second 1802 portions of the graph1800. The first object 1802 is traveling at a higher speed as can bediscerned from the higher frequency of the pulses of graph 1800 in thetime interval t=400 to t=500. The second object, however, is travelingat a slower speed than the first object based on the lower frequency ofthe graph 1800 in the time interval t=700 to t=900. A lower frequencyimplies that it takes more time for the second object to pass the samemask feature lengths as the first object. Since the velocity differenceis considerably larger than in FIG. 17, the two objects are going in andout of phase much more rapidly, hence the pattern of non-zero negativepeaks is more complex, containing not one but several out-of-phaseregions.

FIG. 19 illustrates a graph 1900 of the time varying detector signalhaving a shape indicative of three objects of approximately the samelength traveling in close proximity in the detection region. The firstobject enters the detection region first at about time t=50. The othertwo objects are overlapping in the detection region and followingclosely behind the first object, starting at about time t=400. Thepresence of the first object that partially overlaps at least the secondobject is indicated by portion 1903 of graph 1900. The presence of thesecond and third objects that are partially overlapping is indicated byportions 1901, 1902 of graph 1900, respectively. Light emanating fromthe first, second, and third objects produces separate peaks 1905, 1906,1907 of the upper modulation envelope. The constructive interferencepattern of going in and out of phase between the second and thirdobjects of portions 1901 and 1902 is similar to the situation if FIG.18, with the exception that the object 1902 is traveling at a lowervelocity than 1901 (the reverse of FIG. 18). In addition, the overlapsituation between the first object 1903 and pair of overlapping objects1902 and 1901 is similar to that in FIG. 17 except that one object inFIG. 17 is now replaced with a pair of overlapping object in FIG. 19.

The use of a spatial filter can provide the ability to accuratelymeasure object length and velocity using a single detector in a highthroughput cytometry settings. The system can tell, based on theresulting waveforms exactly how many objects are traveling in thechannel, whether each object is accelerating or slowing down, and howmany objects overlap and by how much. In consequence, the system may beable to much more accurately count how many objects have truly passed inthe detection region, including overlapping objects, and provide robustinformation about each object length and velocity. In contrast, existingsystems that lack a spatial filter mask as disclosed herein are prone tounderestimating the number of objects by counting only one objectinstead of two or more in cases of collisions (i.e., multiple particlesin the detector region). Furthermore, the knowledge of each objectlength and velocity can be used to eliminate objects outside the rangeof interest, for example objects that are too large or too small (interms of the length), or traveling at too high or too slow speeds, etc.,which could not be members of the particular objects of interest (e.g.,a particular bacteria species, or beads of certain size).

In some implementations, the velocity of the objects can be determinedby calculating an average of the positive and negative going pulsewidths in the pulse pairs. The velocity of the object is related to theslope of the averages with respect to a pulse (or mask feature) number.

Although the subject matter has been described in language specific tostructural features and/or methodological acts, it is to be understoodthat the subject matter defined in the appended claims is notnecessarily limited to the specific features or acts described above.Rather, the specific features and acts described above are disclosed asrepresentative forms of implementing the claims.

1. A system, comprising: a spatial filter having a plurality of maskfeatures; at least one detector positioned to sense light emanating fromat least one object moving in a flow path along a flow direction, anintensity of the sensed light being time modulated according to the maskfeatures, the detector configured to generate a time varying electricalsignal comprising a sequence of pulses in response to the sensed light;and an analyzer configured to measure a pulse width of at least some ofthe pulses at a predetermined fraction of an amplitude extremum of thepulses and to determine a length of the object along the flow directionbased on the measured pulse widths.
 2. The system of claim 1, whereinthe first features are substantially transparent and the second featuresare substantially opaque.
 3. The system of claim 1, wherein the analyzeris configured to: determine a function, f({p_(i)},x) that fits a set ofmask feature measurement points {p_(i)}, each point given byp_(i)=(x_(i),y_(i)), where each x_(i) is associated with a featurelength of an i^(th) mask feature and each y_(i) is associated with ani^(th) measured pulse width associated with the i^(th) mask feature, thefunction predicting an expected pulse width measurement of ahypothesized mask feature of length x based on the set of mask featuremeasurement points {p_(i)}.
 4. The system of claim 3, wherein thefunction is a linear function.
 5. The system of claim 4, wherein: thefirst mask features are substantially optically transparent and thesecond mask features are substantially optically opaque; andextrapolating the object length using the function comprises determiningf({p_(i)}, for x=0).
 6. The system of claim 5, wherein the mask featuresare arranged so that a length of the mask features changes linearlyalong the flow direction.
 7. The system of claim 5, wherein the maskfeatures are arranged so that a length of the mask features changesnon-linearly along the flow direction.
 8. The system of claim 3, whereinthe function is an exponential or logarithmic function.
 9. The system ofclaim 8, wherein the mask features are arranged so that the length ofthe mask features changes logarithmically along the flow direction. 10.The system of claim 1, wherein a frequency of the mask features isconstant along the flow direction and corresponds to a fixed pitchvalue.
 11. The system of claim 1, wherein the fraction of the pulseextremum is in a range of about 10% to 40% or about 60% to 90%.
 12. Thesystem of claim 1, wherein: the mask features include a first sectionarranged in a first linear chirp or logarithmic chirp pattern and asecond section arranged in a second linear or logarithmic chirp pattern,wherein the first pattern and the second pattern are symmetrical arounda center line extending laterally across the spatial filter; the firstmask features are substantially transparent and the second mask featuresare substantially opaque and the transparent features have a firstspecified length proximate to the center line; and the transparentfeatures of the first pattern have a linear decrease in length of asecond specified value along the flow direction and the transparentfeatures of the second pattern have a linear increase in length of thesecond specified value along the flow direction.
 13. The system of claim1, further comprising a light source configured to provide input light,wherein the light emanating from the objects is responsive to the inputlight.
 14. The system of claim 1 wherein the intensity distribution ofthe input light is approximately Gaussian or Lambertian.
 15. The systemof claim 1, wherein: the mask features include first features and secondfeatures, a length of the first features is constant along the flowdirection of the spatial filter and a length of the second features isconstant along the flow direction of the spatial filter; the analyzer isconfigured to: measure pulse widths of positive going pulses; measurepulse widths of negative going pulses; and determine the length of theobject based on averages of widths of pulse pairs, each pulse paircomprising a positive pulse and an adjacent negative pulse.
 16. Thesystem of claim 15, wherein the analyzer is further configured todetermine whether a velocity of the object is increasing or decreasingbased on a slope of the averages of the pulse width pairs.
 17. Thesystem of claim 1, wherein: a length of the first features is constantalong the flow direction of the spatial filter; a length of the secondfeatures is constant along the flow direction of the spatial filter; theat least one object comprises multiple objects; the detector ispositioned to sense light emanating from the multiple objects moving ina flow path along the flow direction; the analyzer is configured to:measure pulse widths of positive going pulses; measure pulse widths ofnegative going pulses; and identify a number of the multiple objectstraveling together along the flow path based on the pulse widths of thepositive pulses and the negative pulses.
 18. The system of claim 17,wherein the analyzer is configured to determine a distance between twoor more objects traveling together based on the pulse widths of thepositive pulses and the negative pulses.
 19. The system of claim 1,wherein the analyzer is configured to determine an instantaneousvelocity of the object as the object travels along the flow pathrelative to the mask features based on the measured pulse widths. 20.The system of claim 1, wherein the analyzer is configured to determine,on a mask feature-by-feature basis, whether the object is speeding up orslowing down as the object travels along the flow path based on themeasured pulse widths.
 21. A method, comprising: sensing light emanatingfrom at least one object moving in a flow path along a flow direction ofa spatial filter, the spatial filter having a plurality of mask featurescomprising first features alternating with second features along theflow direction, the first features having first light-transmissioncharacteristics and the second features having second light transmissioncharacteristics, different from the first light transmissioncharacteristics, an intensity of the sensed light being time modulatedaccording to the mask features; generating a time varying electricalsignal comprising a plurality of time modulated pulses in response tothe sensed light; measuring pulse widths of the pulses at apredetermined fraction of a maximum extremum of the pulses; anddetermining a length of the object along the flow direction based on themeasured pulse widths.
 22. The method of claim 21, wherein determiningthe length of the object comprises: determining a function, f({p_(i)},x)that fits the set of mask feature measurement points {p_(i)}, each pointgiven by p_(i)=(x_(i),y_(i)), where each x_(i) is associated with afeature length of an i^(th) mask feature and y_(i) is associated with ani^(th) measured pulse width associated with the i^(th) mask feature; thefunction predicting an expected pulse width measurement of anhypothesized mask feature of length x based on the mask featuremeasurement points {p_(i)}; and extrapolating the object length usingthe function.
 23. The method of claim 22, further comprising removingoutliers in measurement points {p_(i)} before determining the function,the outliers corresponding to erroneous measurements.
 24. The method ofclaim 22, wherein extrapolating the object length using the functioncomprises determining f({p_(i)}, for x=0).
 25. The method of claim 21,wherein: a length of the first features is constant along the flowdirection of the spatial filter; a length of the second features isconstant along the flow direction of the spatial filter; measuring thepulse width of each of the pulses comprises: measuring pulse widths ofpositive pulses; measuring pulse widths of negative pulses; anddetermining the length of the object along the flow direction comprisesdetermining the length of the object based on averages of widths ofpulses in pulse pairs, each pulse pair comprising a positive pulse andan adjacent negative pulse.
 26. The method of claim 21, wherein: the atleast one object comprises multiple objects; sensing the emanating lightcomprise sensing light emanating from the multiple objects; measuringthe pulse widths comprises: measuring pulse widths of positive goingpulses; measuring pulse widths of negative going pulses; and identifyinga number of the multiple objects traveling together along the flow pathbased on the pulse widths of the positive going pulses and the negativegoing pulses.
 27. The method of claim 26, further comprising determininga distance along the flow direction between two or more objectstraveling at a velocity based on the pulse widths of the positive pulsesand the negative pulses.
 28. The method of claim 21, further comprisingdetermining an instantaneous velocity of the object as the mask movesalong the flow path past the mask features based on the measured pulsewidths.
 29. A system, comprising: a spatial filter having a plurality ofmask features; at least one detector positioned to sense light emanatingfrom at least one object moving in a flow path along a flow direction,an intensity of the sensed light being time modulated according to themask features, the detector configured to generate a time varyingelectrical signal comprising a sequence of pulses in response to thesensed light; and an analyzer configured to measure a pulse width of atleast some of the pulses at in a range of about 10% to 40% or 60% to 90%of an amplitude extremum of the pulses and to determine, based on thepulse widths, one or more of: a length along the flow direction of theobject; an instantaneous velocity of the object; whether the object isaccelerating or decelerating.